Académique Documents
Professionnel Documents
Culture Documents
Food Science and Human Nutrition, Center for Crops Utilization Research,
Iowa State University, Ames, Iowa 50011; telephone: (515)-294-3157; fax:
515-294-6261; e-mail: zivko@iastate.edu
2
Department of Agricultural and Biosystems Engineering, Iowa State
University, Ames, Iowa
3
ProdiGene, Inc., College Station, Texas
Received 13 January 1997; accepted 18 April 1997
INTRODUCTION
Recombinant protein production systems utilized today
range from prokaryotic systems such as Escherichia coli
(Georgiou, 1988; Georgiou and Bowden, 1991; Kudo,
1994) and Bacillus (Ebisu et al., 1996; Udaka and
Yamagata, 1993), to eukaryotic systems such as yeast (Ha-
CCC 0006-3592/97/050473-12
duction systems. This review, based on our recent experience in producing the first commercial recombinant proteins
in plants, brings forward a whole set of issues to be tackled
in the process of selecting and developing a winning transgenic plant system.
OPTIMIZATION OF PROTEIN INTEGRITY
AND ACCUMULATION
Historical Perspective
Since the first transgenic plant was introduced in 1983 (Fraley et al., 1983; Zambryskyi et al., 1983), considerable effort has been directed to developing reliable transformation
systems and expression vectors that permit high levels of
gene expression and to direct recombinant proteins to specific plant tissues. Recombinant proteins from a wide variety of sources were produced in transgenic plants such as
tobacco, tomato, petunia, potato, corn, alfalfa, canola, and
soybeans, to name a few. Proteins with molecular weights
as small as 0.6 kD (Turpen et al., 1995) and as large as 80
kD (Verwoerd et al., 1995) per subunit have been produced
(Table I). Although reported accumulation levels are difficult to compare because different plant expression systems
Recombinant
protein
Molecular
weight
(kD/subunit)
Origin
Host
Production level
Reference
a-Amylase
Aprotinin
55.2
6
Bacillus licheniformis
Bovine
Tobacco
Corn
Avidin
Chymosin
15
30
Chicken
Calf
Cyclodextrin
glucanotransferase
Enkephalin
Erythropoietin
Glucoamylase
b-Glucuronidase
76
Klebsiella pneumoniae
Corn
Tobacco,
potato
Potato
0.5
37
74
68
Human
Human
Aspergillus niger
Escherichia coli
Arabidopsis
Tobacco
Potato
Corn
Growth hormone
Heat-labile
enterotoxin B
Hepatitis B
surface antigen
Hirudin
g- and k-chains
hybridoma
b-Interferon
Levansucrase
21
39
Trout
Escherichia coli
24
Hepatitis B virus
Tobacco
Tobacco,
potato
Tobacco
11
45 and 27
Hirudo medicinalis
Mouse
Canola
Tobacco
1% of seed weight
1.3% of soluble leaf protein
20
49.9
Human
B. subtilis, Streptococcus
mutans
Chicken
Plasmodium
Aspergillus niger
Castor bean
Human
Clostridium thermocellum
Tobacco
Tobacco,
potato
Tobacco
Tobacco
Tobacco
Tobacco
Potato
Tobacco
Lysozyme
Malarial epitopes
Phytase
Ricin
Serum albumin
Xylanase
474
14.4
0.6
80
34
66.5
37
475
can affect the expression levels include pre-mRNA processing (capping, splicing, and polyadenylation) and transcript
stability. Introns occur naturally in many eukaryotic genomic DNAs (Lambowitz and Belfort, 1993) and must be
spliced from a pre-mRNA nucleic acid. Processing of the
RNA depends on the accurate recognition of the splice sites
and the proper splicing machinery (Brown et al., 1993; Luehrsen et al., 1994; Solymosy, 1990; Solymosy and Pollak,
1993). The introduction of genes into plants by rDNA methods has demonstrated that introns can actually increase the
stability of the RNA (Topfer et al., 1993), which in turn
generates a dramatic increase in the ultimate level of protein
produced in the transgenic plants. The use of introns has
been particularly successful in elevating the levels of expression in monocots such as maize (Callis et al., 1987;
Maas et al., 1991).
Polyadenylation sites also strongly influence the stability
of the message and, ultimately, the level of gene expression
in plant cells (Hunt, 1994; Ingelbrecht et al., 1989). In addition, there are specific recognition sites that enhance RNA
decay (Sullivan and Green, 1993). Some of these sites have
been identified and are thought to correspond to specific
binding sites (Taylor and Green, 1995). To increase the
accumulation of recombinant protein, it may be necessary to
screen for these sites and modify the gene to remove them
whenever possible. A significant effort has been made in the
last few years to better understand stability and decay
mechanisms of plant mRNA. The current knowledge of the
factors affecting mRNA stability has been summarized by
Abler and Green (1996).
Translation
Targeting
476
Posttranslational Processing
Posttranslational modifications can have dramatic effects on
the end product, but detailed analysis has been omitted in
most studies dealing with the expression of plant genes.
There are examples that demonstrate that properly processed recombinant proteins can be produced in plants, as
well as examples of incomplete processing (Table II). Because this aspect is critical to the integrity (quality) of the
final product, a discussion of some of the issues and representative examples is appropriate.
Table II. Pre- and prosequences used for recombinant protein production in transgenic plants.
Recombinant protein
a-Amylase
a-Amylase
a-Zein
b-Zein
Hybridomas of gand k-chains
2S Albumin
Chymotrypsin
inhibitor I
CryIA(c)
Enkephalin2S
albumin fusion
Lectin
Legumin
Origin
Host
Presequence
Prosequence
Processing
Bacillus
licheniformis
B. licheniformis
Corn
Corn
Mouse
Tobacco
a-amylase
Proper
Tobacco
Tobacco
Tobacco
Tobacco
Tobacco PR-S
a-Zein
b-Zein
Native
Proper
Proper
Proper
Proper
Brazil nut
Tomato
Tobacco, canola
Nightshade, tobacco,
alfalfa
Tobacco
Canola
Native
Native
Native
Native
Proper
Proper
Modified ats1A
at2S1
at2S1
Proper
Proper
Potato
Nicotinia
plumbaginafolio
Tobacco
Tobacco,
Brassica napus,
A. thaliana
Tobacco
Potato
Potato
Tobacco
Tobacco
Potato
Native
Proper
a-Zein
AT2S1
Proper
Proper
Native
Native
Native
Native
Human serum
albumin
Native
Proper
Incomplete
Incomplete
Proper
Proper
Improper
Incomplete
B. thuringiensis
Pisum sativum
Pisium salivus
Modified a-zein
Modified 2S albumin
Brazil nut
Modified glycinin
Modified proglycinin
Native proglycinin
Normal glycinin
Phytase
Pro-serum albumin
Soybean
Soybean
Aspergillus niger
Human
Prohevein
Hevea brasiliensis
(rubber tree)
Human
Barley, wheat
Tomato
Native
Native
Native
Native
Tobacco PR-S
Human serum
albumin
Potato
Tobacco
Tobacco PR-S
Native
No prosequence
Native
Proper
Proper
Fish
Tobacco
Native
Native
Proper, but
not efficient
Serum albumin
Thionin
Type 1 fish
antifreeze protein
Reference
Proteolytic Processing
The outcome of proteolytic processing of various recombinant proteins produced in transgenic plants is summarized
in Table II. In several instances, recombinant proteins were
not properly cleaved, which resulted in the accumulation of
pro-proteins instead of mature proteins. Normal and modified prepro-glycinins were not correctly processed in tubers
of transgenic potato (Utsumi et al., 1994), but were correctly
processed in seeds, leaves, and stems of transgenic tobacco
(Utsumi et al., 1993). The incorrect processing of the recombinant glycinins in potato tubers indicates that the vacuoles of potato tubers lack the enzymes necessary for proteolytic cleavage of proglycinins to mature glycinins
(Utsumi et al., 1994). Similar to the processing of the normal and modified prepro-glycinins, prepro-human serum
albumin (HSA) was only processed to pro-HSA in tobacco
and potato. The apparent problem that stemmed from the
lack of serine proteases in the plant has been resolved by
directly fusing the HSA to the signal sequence of the tobacco P-RS protein. This fusion allowed the formation of
mature HSA indistinguishable from the authentic human
protein (Sijmons et al., 1990).
Tobacco and Brassica napus have successfully performed a several-step proteolytic processing of recombinant
thionins (Carmona et al., 1993; Florack et al., 1994), Brazil
nut 2S albumin (Altenbach et al., 1989, 1992) and human
protein C (Cramer et al., 1996). The cleavage of both the
aminoterminal signal peptide and the carboxyterminal peptide of prepro-thionins appeared to be important for mediating the transition of the recombinant protein into the endoplasmic reticulum and the correct folding of mature thionins, respectively (Florack et al., 1994).
In summary, plants possess the machinery necessary for
required proteolytic processing, but not all plant tissue and
gene combinations give the same results as the native host.
To take full advantage of plant systems more work is
needed to develop strategies for achieving proper processing.
Glycosylation
One of the advantages of the plant production systems is the
ability to perform posttranslational modifications such as
protein glycosylation. There are only a few studies that have
477
478
Protein Degradation
The level of protein accumulation is the consequence of not
only the synthesis of the protein but the degradation as well.
There are known systems which aid in protein degradation
such as the ubiquitin system (Hondred and Vierstra, 1992;
Huffaker, 1990; Parsell and Lindquist, 1993; Vierstra,
1993). In general, it is believed that smaller peptide sequences will degrade faster. There have also been examples
that have demonstrated that recombinant proteins can be
degraded at rates which result in a significantly reduced
level of the end product in the cell (Hoffman et al., 1988;
Ohtani et al., 1991; Utsumi et al., 1993).
There are several potential ways to reduce protein degradation: (1) to express the recombinant protein in seed
tissues where levels of protease inhibitors may slow the rate
of protein degradation; (2) to target the protein to the endoplasmic reticulum (Fiedler and Conrad, 1995; Wandelt et
al., 1992); (3) to engineer proteins without protease-specific
Figure 1. Cost of producing a recombinant protein assuming accumulation of 10% (w/w) of the total crop protein.
479
480
gether with other seed or tissue proteins to determine accumulation levels, to establish protein functionality, and to
confirm the expected amino acid sequence. Depending on
the transgenic plants and the nature of the recombinant protein, different extraction solutions were used. Beyond control of ionic strength and pH, extraction has been aided by
use of detergents (Mason et al., 1992, 1996; Sehnke et al.,
1994; Thanavala et al., 1995), reducing agents (Fuchs et al.,
1993; Voelker et al., 1989), and protease inhibitors such as
phenylmethanesulphonlyl fluoride (PMSF) (Hiatt et al.,
1989; Sijmons et al., 1990; Mason et al., 1992, 1996; Thanavala et al., 1995) or protease inhibitor mixtures containing
PMSF, leupeptin, and benzamidine (Fuchs et al., 1993).
Unfortunately, the purification of recombinant proteins produced in transgenic plants has been performed mainly on a
laboratory scale using methods not differing greatly from
those traditionally used for isolation of plant proteins (Fuchs
et al., 1993; Hood et al., 1997; Kusnadi et al., 1997; Mason
et al., 1992, 1996; Ohtani et al., 1991, Sijmons et al., 1990;
Vandekerckhove et al., 1989). Most laboratory-developed
procedures may not be directly scalable because of the high
cost of chemicals, the difficulties in controlling the process
shear and foaming, and the altered characteristics of the
processed transgenic material (particle size, moisture content, etc.) caused by the differences between the laboratory
and plant equipment.
The feasibility for recovering recombinant enzymes from
transgenic alfalfa has been evaluated recently by Austin et
al. (1994). The recovery steps included juice extraction by
maceration, clarification, concentration, and stabilization of
the soluble protein concentrate. Further fractionation and
purification was not investigated but losses of enzymatic
activity in the extract were observed. We have compared the
laboratory-scale extraction of recombinant b-glucuronidase
(GUS) from transgenic maize, soybeans, and canola seeds
and determined that, except for canola (40% oil content),
the presence of oil and/or starch in the ground seed samples
did not affect the extraction yield (unpublished data). The
extraction was carried out only with 0.05 M phosphate
buffer (pH 7.5) and no proteolytic degradation was observed. Further purification of GUS by ion-exchange and
hydrophobic interaction chromatography was not affected
by the soluble starch or corn oil in the clarified extract
(Kusnadi et al., 1997). Similarly, the extraction and affinity
purification yields of recombinant avidin from transgenic
maize were not significantly affected by the complex maize
extract (Hood et al., 1997).
Transgenic plants offer unique opportunities to simplify
recombinant protein extraction and purification. One avenue worth exploring is the targeting of the recombinant
protein synthesis to a tissue or organelle that can be easily
separated from the rest of the plant material. For example,
by using the maize germ (10% of the seed) as a starting
material for the extraction, we have increased the concentration of GUS in the extract almost ten times, because 90%
of the extractable enzyme activity was located in the germ.
Recently, Van Rooijen and Moloney (1995) demonstrated
References
Abler, M. L., Green, P. J. 1996. Control of mRNA stability in higher
plants. Plant Mol. Biol. 32: 6378.
Allen, G. C., Hall, G., Jr., Michalowski, S., Newman, W., Steven, S.,
Weissinger, A. K., Thompson, W. F. 1996. High-level transgene expression in plant cells: effects of a strong scaffold attachment region
from tobacco. Plant Cell 8: 899913.
Altenbach, S. B., Kuo, C. C., Staraci, L. C., Pearson, K. W., Wainwright,
C., Georgescu, A., Townsend, J. 1992. Accumulation of a Brazil nut
albumin in seeds of transgenic canola results in enhanced levels of
seed protein methionine. Plant Mol. Biol. 18: 235245.
Altenbach, S. B., Pearson, K. W., Meeker, G., Staraci, L. C., Sun, S. S. M.
1989. Enhancement of the methionine content of seed proteins by the
expression of a chimeric gene encoding a methionine-rich protein in
transgenic plants. Plant Mol. Biol. 13: 513522.
Archer, D. B. 1994. Enzyme production by recombinant Aspergillus, pp.
373393. In: Y. Murooka and T. Amanaka (eds.), Recombinant microbes for industrial and agricultural applications. Marcel Dekker,
New York.
Austin, S., Bingham, E. T., Koegel, R. G., Mathews, D. E., Shahan, M. N.,
Straub, R. J., Burgess, R. R. 1994. An overview of a feasibility study
for the production of industrial enzymes in transgenic alfalfa. Ann. NY
Acad. Sci. 721: 235244.
Beachy, R. N., Chen, Z. L., Horsch, R. B., Rogers, S. G., Hoffmann, N. J.,
Fraley, R. T. 1985. Accumulation and assembly of soybean bconglycinin in seeds of transformed petunia plants. EMBO J. 4:
30473053.
Bednarek, S. Y., Raikhel, N. V. 1992. Intracellular trafficking of secretory
proteins. Plant Mol. Biol. 20: 133150.
Benfey, P. H., Chua, N. H. 1989. Regulated genes in transgenic plants.
Science 244: 174181.
Bosch, D., Smal, J., Krebbers, E. 1994. A trout growth hormone is expressed, correctly folded and partially glycosylated in the leaves but
not the seeds of transgenic plants. Transgen. Res. 3: 304310.
Boulikas, T. 1994. Myb proteins talking to their DNA (review). Int. J.
Oncol. 5: 101109.
Brown, S. M., Santino, C. G. 1995. Enhanced expression in plants, U.S.
patent 5,424,412.
Brown, J. W. S., Simpson, C. G., Simpson, G. G., Turnbull-Ross, A. D.,
Clark, G. P. 1993. Plant pre-mRNA splicing and splicing components.
Phil. Trans. Roy. Soc. Lond. B 342: 217224.
Bruyns, A.-M., de Jaeger, G., de Neve, M., de Wilde, C., van Montagu, M.,
Depicker, A. 1996. Bacterial and plant-produced scFv proteins have
similar antigen-binding properties. FEBS Lett. 386: 510.
Callis, J., Fromm, M., Walbot, V. 1987. Introns increase gene expression
in cultured maize cells. Genes Devel. 1: 11831200.
Carmona, M. J., Molina, A., Fernandez, J. A., Lopez-Fando, J. J., GarciaOlmedo, F. 1993. Expression of the a-thionin gene from barley in
tobacco confers enhanced resistance to bacterial pathogens. Plant J. 3:
457462.
Cavener, D. R., Ray, S. C. 1991. Eukaryotic start and stop translation sites.
Nucl. Acids Res. 19: 31853192.
Chrispeels, M. J. 1991. Sorting of proteins in the secretory system. Annu.
Rev. Plant Physiol. Plant Mol. Biol. 42: 2153.
Cornelissen, M., Ellebogten, H., Soetaert, P., Eekhoekstraat, L., Stam, M.,
Uilenstede, A. A., Dockx, J., Tentoonstellingslaan, G., Van Aarssen,
R., Zwigjnaardsesteenweg, G. 1993. Modified genes and their expression in plant cells, international patent WO 93/09218.
Cramer, C. L., Weissenborn, D. L., Oishi, K. K., Grabau, E. A., Bennet, S.,
Ponce, E., Grabowski, G. A., Radin, D. N. 1996. Bioproduction of
481
482
soluble and secreted forms of human parainfluenza virus type 3 glycoproteins expressed from mammalian and insect cells as subunit vaccines. J. Gen. Virol. 74: 459469.
Lesser, E. W., Asenjo, J. A. 1992. Rational design of purification processes
for recombinant proteins. J. Chromatogr. 584: 4357.
Lorenz, K. J., Kulp, K. 1991. Handbook of cereal science and technology.
Marcel Dekker, New York.
Lubon, H., Paleyanda, R. K., Velander, W. H., Drohan, W. N. 1996. Blood
proteins from transgenic animal bioreactors. Transufs. Med. Rev. 10:
131143.
Luckow, V. A., Summers, M. D. 1988. Trends in the development of baculovirus expression vectors. Bio/Technology 6: 4755.
Luehrsen, K. R., Taha, S., Walbot, V. 1994. Nuclear pre-mRNA processing
in higher plants. Prog. Nucl. Acid Res. Mol. Biol. 47: 149193.
Lutcke, H. A., Chow, K. C., Mickel, F. S., Moss, K. A., Kern, H. F.,
Scheele, G. A. 1987. Selection of AUG initiation codons differs in
plants and animals. EMBO J. 6: 4348.
Ma, J. K.-C., Hein, M. B. 1995. Immunotherapeutic potential of antibodies
produced in plants. Trends Biotechnol. 13: 522527.
Ma, J. K.-C., Hiatt, A., Hein, M., Vine, N. D., Wang, F., Stabila, P., van
Dolleweerd, C., Mostov, K., Lehner, T. 1995. Generation and assembly of secretory antibodies in plants. Science 268: 716719.
Maas, C., Laufs, J., Grant, S., Korfhage, C., Werr, W. 1991. The combination of a novel stimulatory element in the first exon of the maize
Shrunken-1 gene with the following intron 1 enhances reporter gene
expression up to 1000-fold. Plant Mol. Biol. 16: 199207.
Marino, M. M. 1991. Expression of heterologous proteins in yeast, pp.
2965. In: A. Prokop, R. K. Bajpal, and C. S. Ha (eds.), Recombinant
DNA technology and applications. McGraw-Hill, New York.
Mason, H. S., Ball, J. M., Shi, J.-J., Jiang, X., Estes, M. K., Arntzen, C. J.
1996. Expression of Norwalk virus capsid protein in transgenic tobacco and its oral immunogenicity in mice. Proc. Natl. Acad. Sci. USA
93: 53355340.
Mason, H. S., Lam, D. M.-K., Arntzen, D. J. 1992. Expression of hepatitis
B surface antigen in transgenic plants. Proc. Natl. Acad. Sci. USA 89:
1174511749.
Matsumoto, S., Ikura, K., Ueada, M., Sasaki, R. 1995. Characterization of
a human glycoprotein (erythropoietin) produced in cultured tobacco
cells. Plant Mol. Biol. 27: 11631172.
Matthews, B. F., Hughes, C. A. 1993. Nutritional improvement of the aspartate family of amino acids in edible crop plants. Amino Acids 4:
2134.
Meyer, P., Saedler, H. 1996. Homology-dependent gene silencing in plants.
Annu. Rev. Plant Physiol. Plant Mol. Biol. 47: 2348.
Murray, E. E., Lotzer, J., Eberle, M. 1989. Codon usage in plant genes.
Nucl. Acids Res. 17: 477498.
Narvaez-Vasquez, J., Orozco-Cardenas, M. L., Ryan, C. A. 1992. Differential expression of a chimeric CaMV-tomato proteinase inhibitor I
gene in leaves of transformed nightshade, tobacco and alfalfa. Plant
Mol. Biol. 20: 11491157.
Oakes, J. V., Shewmaker, C. K., Stalker, D. M. 1991. Production of cyclodextrins, a novel carbohydrate, in the tubers of transgenic potato
plants. Bio/Technology 9: 982986.
Ohtani, T., Galili, G., Wallace, J. C., Thompson, G. A., Larkins, B. A.
1991. Normal and lysine-containing zeins are unstable in transgenic
tobacco seeds. Plant Mol. Biol. 16: 117128.
Park, Y.-D., Papp, I., Moscone, E. A., Iglesias, V. A., Vaucheret, H.,
Matzke, A. J. M., Matzke, M. A. 1996. Gene silencing mediated by
promoter homology occurs at the level of transcription and results in
meiotically heritable alterations in methylation and gene activity. Plant
J. 9: 183194.
Parmenter, D. L., Boothe, J. G., van Rooijen, G. J. H., Yeung, E. C.,
Moloney, M. M. 1995. Production of biologically active hirudin in
plant seeds using oleosin partitioning. Plant Mol. Biol. 29: 11671180.
Parsell, D. A., Lindquist, S. 1993. The function of heat-shock proteins in
stress tolerance: degradation and reactivation of damaged proteins.
Annu. Rev. Gen. 27: 437496.
Pen, J., Molendijk, L., Quax, W. J., Sijmons, P. C., van Ooyen, A. J. J., van
483
Trudel, J., Potvin, C., Asselin, A. 1992. Expression of active hen egg white
lysozyme in transgenic tobacco. Plant Sci. 87: 5567.
Turpen, T. H., Reinl, S. J., Charoenvit, Y., Hoffman, S. L., Fallarme, V.,
Grill, L. K. 1995. Malarial epitopes expressed on the surface of recombinant tobacco mosaic virus. Bio/Technology 13: 5357.
Udaka, S., Yamagata, H. 1993. High-level secretion of heterologous proteins by Bacillus brevis. Meth. Enzymol. 217: 2333.
Utsumi, S., Kitagawa, S., Katsube, T., Higasa, T., Kito, M., Takaiwa, F.,
Ishige, T. 1994. Expression and accumulation of normal and modified
soybean glycinins in potato tubers. Plant Sci. 102: 181188.
Utsumi, S., Kitagawa, S., Katsube, T., Kang, I. J., Gidamis, A. B.,
Takaiwa, F., Kito, M. 1993. Synthesis, processing and accumulation of
modified glycinins of soybean in the seeds, leaves and stems of transgenic tobacco. Plant Sci. 92: 191202.
Van den Elzen, P. J. M., Pen, J., Hoekema, A., Sijmons, P. C., Van Ooyen,
A. J. J., Rietveld, K., Quax, W. J. 1992. Transgenic plants having a
modified carbohydrate content, international patent WO 92/05259.
Van der Meer, I. M., Ebskamp, M. J. M., Visser, R. G. F., Weisbeek, P. J.,
Smeekens, S. C. M. 1994. Fructan as a new carbohydrate sink in transgenic potato plants. Plant Cell 6: 561570.
Vandekerckhove, J., Damme, J. V., Lijsebettens, M. V., Botterman, J.,
Block, M. D., Vandewiele, M., Clercq, A. D., Leemans, J., Montagu,
M. V., Krebbers, E. 1989. Enkephalins produced in transgenic plants
using modified 2S seed storage proteins. Bio/Technology 7: 929932.
Van Rooijen, G. J. H., Moloney, M. M. 1995. Plant seed oil-bodies as
carriers for foreign proteins. Bio/Technology 13: 7277.
Velander, W. H., Lubon, H., Drohan, W. N. 1997. Transgenic livestock as
drug factories. Sci. Am. 276: 7074.
Verwoerd, T. C., van Paridon, P. A., van Ooyen, A. J. J., van Lent,
J. W. M., Hoekema, A., Pen, J., 1995. Stable accumulation of Aspergillus niger phytase in transgenic tobacco leaves. Plant Physiol. 109:
11991205.
Vierstra, R. D. 1993. Protein degradation in plants. Annu. Rev. Plant Physiol. Plant Mol. Biol. 44: 385410.
484
Vitale, A., Ceriotti, A., Denecke, J. 1993. The role of the endoplasmic
reticulum in protein synthesis, modification, and intracellular transport. J. Exp. Botany 44: 14171444.
Voelker, T. A., Herman, E. M., Chrispeels, M. J. 1989. In vitro mutated
phytohemagglutinin genes expressed in tobacco seeds: role of glycans
in protein targeting and stability. Plant Cell 1: 95104.
Wandelt, C. I., Khan, M. R. I., Craig, S., Schroeder, H. E., Spencer, D.,
Higgins, T. J. V. 1992. Vicilin with carboxy-terminal KDEL is retained in the endoplasmic reticulum and accumulates to high levels in
the leaves of transgenic plants. Plant J. 2: 181192.
Warren, T. G., Krivi, G. G. 1991. Strategies for production of proteins in
mammalian cells, pp. 6696. In: A. Prokop, R. K. Bajpai, and C. S. Ho
(eds.), Recombinant DNA technology and applications. McGraw-Hill,
New York.
Werner, R. G., Thomae, K. 1994. Successful products and future business
prospects, pp. 573578. In: R. E. Spier, J. B. Griffiths, and W. Berthold (eds.), Animal cell technology products of today prospects for
tomorrow. Butterworth-Heinemann, Oxford.
Wheelwright, S. M. 1991. In: Protein purification: design and scale up of
downstream processing. Springer, New York.
Whitelam, G. C., Cockburn, B., Gandecha, A. R., Owen, M. R. L. 1993.
Heterologous protein production in transgenic plants. Biotechnol.
Genet. Eng. Rev. 11: 129.
Willmitzer, L., Sonnewald, U., Roeber, M., Carlsen, S. K. 1992. Transgenic plants expressing genes for industrial enzymes, international
patent WO 92/01042.
Wong, E. Y., Hironaka, C. M., Fischhoff, D. A. 1992. Arabidopsis
thaliana small subunit leader and transit peptide enhance the expression of Bacillus thuringiensis proteins in transgenic plants. Plant Mol.
Biol. 20: 8193.
Zambryski, P., Joss, H., Genetello, C., Leemans, J., van Montagu, M.,
Schell, J. 1983. Ti plasmid vector for the introduction of DNA into
plant cells without alteration of their normal regeneration capacity.
EMBO J. 2: 21432150.